Sanprep column plasmid mini preps kit

DNA damage protection activities of polysaccharides were determined with pUC19 plasmid DNA, isolated from Escherichia coli DH5α with a SanPrep Column Plasmid Mini-Preps Kit (Sangon Biotech, Shanghai, China). pUC19 plasmid was damaged by H₂O₂ and UV treatment using the method of Yang et al. [31 (link)]. Rutin was used as a positive control. Different structural or conformational forms of plasmid DNA were separated by electrophoresis. The reaction mixture (10 mL) contained 3 mL of plasmid DNA, 5 mL of 5 mg/mL polysaccharide or 0.4 mg/mL rutin, 1 mL of 10 mmol/mL H₂O₂, and 1 mL of water. The mixtures were located in a super clean bench with an ultraviolet lamp (20 W). After UV irradiation lasted for 5 min at room temperature, reaction samples along with a 10× gel loading dye were analyzed on a 1% agarose gel in TBE buffer at pH 8.0 for 30 min (100 V).

Construct maps of the TRV1, TRV2, and TRV2-CYC-B plasmids were shown in Figure 2. Primers with EcoR I and BamH I restriction enzyme cutting sites (underlined) were designed in the conserved region of CYC-B gene sequence (pCYC-B_F: 5′-CGGGATCCATGGAGTTTGGGTTGATGAA-3′ and pCYC-B_R: 5′-CGGAATTCGCCTACTAACAAGCGAAGTT-3′). The fragment of CYC-B gene from ‘Zaozhong 6′ was inserted in an antisense orientation before the nopaline synthase (NOS) terminator and after the cauliflower mosaic virus (CaMV) 35S promoter. CYC-B fragment was purified using a SanPrep Column DNA gel extraction kit (Sangon, Shanghai, China). The purified product and TRV2 plasmid were both digested by EcoR I and BamH I restriction enzymes. After ligating with T4 ligase overnight, it was transferred into DH5α Escherichia coli (E. coli) competent cells. E. coli was spread using selective media containing kanamycin (Kan, 50 mg/L). After expansion, the quality of E. coli was ascertained by running PCR and agarose gel electrophoresis. A pair of detection primer for TRV2-CYC-B (S1_F: 5′-CGGACGAGTGGACTTAGATTCTG-3′ and S1_R: 5′-GCCTATGGCTTCTGTTCATGTG-3′) were synthesized. TRV2-CYC-B plasmid DNA was extracted using a SanPrep Column Plasmid Mini-Preps kit (Sangon, Shanghai, China), as identified by EcoR I and BamH I digestion then verified by sequencing.

Escherichia coli (E. coli) Top10 and plasmid pET-30a (+) (Invitrogen, Shanghai, China) were used as cloning host and vector, respectively. E. coli BL21(DE3) strain (Invitrogen, Shanghai, China) was used for protein expression. A SanPrep Column Plasmid Mini-Preps Kit purchased from Sangon Biotech (Shanghai, China) was used to extract plasmids. Modified Bradford Protein Assay Kit (Sangon, Shanghai, China) was used to measure protein concentration. The molecular marker (Code No. 3595Q) was obtained from Takara (Dalian, China). The glycerin monostearate rich oil (≥78%) was synthesized in our laboratory. Glycerol, linolenic acid (~70%), 4-Nitrophenol solution, pNP-C4 to C18 were obtained from Sigma-Aldrich (Shanghai, China). All other chemicals and reagents used in the study were of analytical grade.

For the epidemiological survey of PSV, a total of 260 clinical samples, including feces, fecal swabs, and intestine samples, were collected between 2020 and 2022 from sows and suckling, post-weaning, and fattening pigs that were asymptomatic or with clinical diarrhea (25 with clinical diarrhea and 5 asymptomatic) from 30 farms in Fujian Province. A description of the studied pig population and the number of collected samples for each farm is shown in Supplementary Table S1. All samples were detected for PSV using real-time PCR. The PSV2020 strain was preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences. M-MuLV First Strand cDNA Synthesis Kit (Code No: B532435), Rapid Competent Cell Preps Kit (One-step, Code No: B529307), SanPrep Column Plasmid Mini-Preps Kit (Code No: B518191), SanPrep Spin Column & Collection Kit (Code No: B515103), and Taq Plus DNA polymerase, 2 × Taqman Fast qPCR Mix (High Rox) (Code No: B639276, BBI) were bought from Sangon Biotech (Shanghai, China). RNA extraction kit (Code No: 9767), PrimeScriptTM RT Master Mix Reverse Transcription Kit (Code No: RR036A), TaKaRa LA Taq^® (Code No: RR02MQ), and pMD 18-T Vector (Code No: 6011) were purchased from TaKaRa Bio Inc. (Daliang, China).

For dsRNA synthesis, the partial cDNA sequence of RaSsp1 target gene was massively amplified using gene-specific primer, and the amplified product was recycled with SanPrep Column DNA gel Recovery Kit (Sangon Biotech, Shanghai, China), and then the recycled fragment was subcloned into a pMD^®18-T Vector. The DH5α competent cell (Sangon Biotech, Shanghai, China) was transferred into a recombinant plasmid. The bacteria were evenly spread onto LB solid ampicillin medium and then cultured in an incubator until the plaque grew at 37 °C. Several single colonies were selected for PCR amplification to screen bacterial fluid containing the recombinant plasmid with target fragment, and then they were cultured in an oscillator at 37 °C and 200 rpm for 14 h. Plasmids were extracted using SanPrep Column Plasmid Mini-Preps Kit (Sangon Biotech, Shanghai, China). The target fragment was amplified with primers T7 promoter at the 5′ end using the plasmid as template. PCR products were extracted by phenol chloroform, and dsRNA was synthesized using purified product as a template, which was also purified with phenol chloroform extraction. The purity, concentration, and quality of dsRNA were confirmed in the same way as RNA detection. For the RNAi experiment, EGFP was selected as a control gene, and dsRNA was generated using the EGFP vector pQBI-polII (Wako, Osaka, Japan).

The plasmid pJMG, carrying the aphVIII gene (paromomycin-resistance cassette) was originally modified from pSI103 (Chlamydomonas Resource Center, U.S.A., https://www.chlamycollection.org) and obtained from Dr. Junmin Pan’s laboratory [28 (link)]. The plasmids pJMG expanded in DH5α E. coli were purified with SanPrep Column Plasmid Mini-Preps Kit (Sangon Biotech, China). DNA fragments carrying the aphVIII gene were digested with restriction enzyme QuickCut EcoRI (Takara, Japan) and extracted by SanPrep Column DNA Gel Extraction Kit (Sangon Biotech, China). All DNA plasmids and fragments were quantified by NanoDrop 2000 (Thermo Scientific, U.S.A.).

Thirty of the full-length ORFs were used for heterologous expression in E. coli BL21 (DE3) (Tiangen, Beijing, China) and subsequent cellulase activity assays. Briefly, the above recombinant plasmid containing each of the genes was obtained using a SanPrep Column Plasmid Mini-Preps Kit (Sangon, Shanghai, China) and was transformed into E. coli BL21 (DE3) by heat shock. Transformants were grown in liquid SOC (1 h at 37 °C), plated onto kanamycin (50 µg ml⁻¹)-containing LB agar plates and grown overnight at 37 °C. The colonies confirmed to harbour recombinant plasmid using PCR as described above and stored in liquid cultures [LB, 20% glycerol, kanamycin (50 µg ml⁻¹)] at − 80 °C.